- 學位論文
類器官2.0－架構類器官與腫瘤微環境以及類器官與類器官間的互動模式
Organoid 2.0 – Tailoring Cross-Talks of Tumor Microenvironments-PDOs and PDOs-PDOs
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摘要
類器官是活體微型組織來自成人的正常或癌性組織，其含有正常或癌症幹/祖細胞，意指其由具有自組織和再生能力的分化或胚胎幹細胞（ESC）或者多功能誘導型幹細胞（iPSC）分化而來。類器官是體外微型組織的系統模式，其培養在3D骨架中並加入具有合適且充足的生長因子之特定培養基。來自癌症患者檢體組織的類器官保留了大部分患者本身的腫瘤異質性，這使癌症類器官成為開發精準醫療和個人化醫療的出色系統模式。結合分子生物學技術（包括用於產生轉殖基因類器官的基因編輯），類器官提供很大的機會去進行癌症研究中的轉譯醫學應用。在此論文中，我們建立了具有轉殖基因mPlum穩定表達的人類結直腸癌（CRC）類器官，搭配實驗室已建立完成的轉殖基因細胞或CRC類器官我們進一步建立了三種共培養系統模式：誘導表達型人類永生化視網膜上皮（hTERT-RPE-1）細胞和CRC類器官共培養系統模式、mPlum穩定表達型和誘導表達型CRC類器官共培養系統模式，以及mPlum和eGFP穩定表達型CRC類器官共培養系統模式。另外我們正在建立彩虹型CRC類器官系統模式以進行細胞譜系追蹤。有趣的是，我們觀察到和Musashi-1 eGFP誘導表達型hTERT-RPE-1細胞共培養的CRC類器官之細胞譜系產生變化。此外在共培養系統模式中，誘導表達型hTERT-RPE-1細胞的型態發生改變。這些數據顯示微環境對細胞與類器官皆會產生影響。另外mPlum、eGFP以及誘導表達型CRC類器官被用於闡明細胞間或類器官間的交流與融合。通過追蹤螢光類器官細胞，也許能回答細胞的自組織和遷移是如何決定組織發育的問題。總結來說，藉由將分子生物學技術的優勢帶入培養的人類類器官，我們提出了一個概念性類器官2.0模式從而擴大其生物醫學應用範圍。它提供了以單細胞為單位監測細胞行為的機會，並且可能提供組織內細胞行為的重要細節以應用於轉譯醫學和再生醫學。
並列摘要
Organoids are living mini-tissues derived from adult normal or cancerous tissues with normal or cancer stem/progenitor cell origins, derived from differentiated or embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) with self-organization and rejuvenation abilities. Organoids are system models of in vitro mini-tissues cultured in a 3D scaffold supplemented with specific culture medium with suitable enriched growth factors. Heterogeneities of organoids derived from cancer patient’s biopsy are largely maintained and it makes cancer organoids as an excellent system model for developing precision and personalized medicine. Combined with molecular biology techniques including gene editing for generating transgenic organoids, it has greatly wide-open the opportunities for the translational applications of cancer research. In this thesis, we presented that establishing a long-term cultured human colorectal cancer (CRC) organoids with transgenic mPlum constitutive expression. Combining with other transgenic cells or CRC organoids which had been established in our lab, we further established three co-culture system models: CRC organoids and inducible hTERT-RPE-1 cells co-culture system model, mPlum and inducible CRC organoids co-culture system model, and mPlum and eGFP CRC organoids co-culture system model. Besides, we are setting up the Rainbow CRC organoids system model for lineage tracing. Interestingly, we observed that the cell lineage of CRC organoids co-culture with Musashi-1 eGFP inducible hTERT-RPE-1 cells changes. Furthermore, the morphology of inducible hTERT-RPE-1 cells transforms in the co-culture system model. These data shows the microenvironment can affect both cells and organoids. Besides, mPlum, eGFP, and inducible CRC organoids were applied for elucidating inter-cell or inter-organoid cross-talks and fusion. By tracing fluorescent organoid cells, it is possible to answer the question how cell-cell self-organization and migration determine tissue development. To sum up, we proposed a conceptual organoid 2.0 model to widen the biomedical application by taking the advantages of molecular biological tools into cultured human organoids. It provides chances to monitor cellular behaviors at single cell resolution, and it may provide ultimate details of intra-tissue cellular behaviors for applications in translational medicine and regenerative medicine.
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